The background description provided herein and throughout the application is for the purpose of generally presenting the context of the disclosure. Work of the presently named inventors, to the extent it is described in this background section, as well as aspects of the description that may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the present disclosure.
With the completion of genome projects for numerous model organisms, including mice, drosophila, and zebrafish, there is now a desire to link genes to structures and functions through systematic phenotyping of these model organisms. The work, called “phenome projects,” is to be built on a foundation of anatomical and morphological information, supplemented with molecular, physiological, and behavioral assays.
Zebrafish, for example, are frequently used to provide non-mammalian model of human disease. Zebrafish offer various advantageous experimental features, including short generational time, fecundity, small size, transparency, and powerful genetic tools. The result is the creation of the “Zebrafish Phenome Project.” The international zebrafish research community plans to generate at least one mutant for each gene, and each mutant will need to be phenotyped at multiple ages.
An ideal anato-morphological imaging assay would provide isotropic three-dimensional (3D) cellular resolution across an entire model organism, allowing for determination of cellular and tissue phenotypes arising from specific changes in the genotype. In order to phenotype thousands of mutants. Clearly, high-throughput processing would be needed.
Conventionally, two-dimensional glass and virtual histology slides are used in cellular-scale phenotyping of model organisms. But, with such techniques it is difficult to achieve true 3D information and high throughput. In classical histology, for example, mechanically sliced sections of fixed and wax-embedded tissue are cut into ˜5 micrometer-thick sections, and stained with two different color dyes, hematoxylin and eosin, which stain different cellular components. From here, it is possible, in principle, to generate 3D representations by virtually stacking images obtained from the serial sections. However, the mismatch between histological slice thickness (typically 5 microns) and in-plane resolution (typically 200-500 nm) makes the generation of satisfactory 3D representations impractical. Moreover, the entire process of sectioning, staining, and imaging physical slices of the specimen is labor intensive. And as such, a truly 3D imaging modality would solve the problem of physically sectioning and virtually reassembling the specimen.
There are 3D imaging modalities, such as confocal microscopy, optical projection tomography, or magnetic resonance imaging, but they either lack the required 1-2 micron spatial resolution (MRI) or lack the ability to image in optically opaque whole specimens.
One modality that potentially offers an appealing combination of spatial resolution, penetration, and high throughput is X-ray computed tomography. Micro CT imaging is relatively fast, provides isotropic 3D images, permits virtual sectioning in any direction, and leaves the specimen intact for future imaging or sectioning.
The natural X-ray contrast of most model organisms is not sufficiently high to allow subtle phenotyping. But recently, a number of investigators have demonstrated that the use of single heavy-metal stains, of the kind used in electron microscopy, can be used to significantly increase contrast. Indeed some have recently made use of heavy-metal staining, along with synchrotron-based micro CT at ˜1.4 micron resolution, to produce high-contrast, cellular resolution 3D images of an intact opaque organism.
Yet, despite the strengths of metal-stained CT imaging, a key remaining advantage of classical histology remains, i.e., the ability to use different color stains that target different cellular components, multiplying the specific biological information encoded in the images, and yielding differentiation of tissue types. The staining of biological tissues with two separate dyes, e.g., blue dye (hematoxylin) for nucleic acids and pink dye (eosin) for proteins, has allowed scientists to discern essentially every cell type. In optical histology, multi-color stains can also target cell membranes, nucleic acids, muscle fibers, and the like. Thus far, however, no one has developed a multiple stain process available for effective 3D phenotype imaging.